www.nature.com/scientificreports
OPEN
received: 01 September 2016 accepted: 11 January 2017 Published: 13 February 2017
Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance Ming-Ling Chang1,2, Yu-sheng Lin3,4, Li-Heng Pao5,6, Hsin-Chih Huang1 & Cheng-Tang Chiu1 The pathophysiological implications of plasminogen activator inhibitor-1 (PAI-1) in HCV infection remain obscure. This prospective study evaluated 669 HCV patients, of whom 536 had completed a course of anti-HCV therapy and had pre-, peri- and post-therapy measurements of various profiles, including PAI-1 levels. Multivariate analysis demonstrated, before anti-HCV-therapy, platelet count and PAI-1-rs1799889 genotype were associated with PAI-1 levels. Among patients with a sustained virological response (SVR, n = 445), platelet count was associated with PAI-1 level at 24 weeks posttherapy. GEE analysis showed that PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes affected PAI-1 levels early and late in therapy, respectively. At 24 weeks post-therapy, higher lipid, brain natriuretic peptide, homocysteine and PAI-1 levels and PAI-1 activity were noted only in SVR patients compared with pre-therapy levels. Within 24 weeks post-therapy, 2.2% of the SVR (mean age: 57.8 yr; 8 smoking males; the 2 females had pre-therapy hypercholesteremia or cardiovascular family history of disease) and 0% of the non-SVR patients experienced a new cardiovascular event. Platelet counts consistently correlated with PAI-1 levels regardless of HCV infection. PAI-1-rs-1799889 and interferonλ3-rs12979860 genotypes mainly affected PAI-1 levels longitudinally. Within 24 weeks post-antiHCV therapy, the SVR patients showed increasing PAI-1 levels with accelerating cardiovascular risk, especially the vulnerable cases. Hepatitis C virus (HCV), a human pathogen responsible for acute and chronic liver disease, has variants that are classified into 7 major genotypes and has infected an estimated 170 million individuals worldwide1. HCV infection is believed to cause metabolic alterations, including steatosis, hypolipidemia, insulin resistance (IR), diabetes and obesity2. Despite the favorable lipid profile that results from HCV infection, many studies have shown that HCV infection unfavorably impacts cardiovascular events after adjusting for conventional risk factors2,3. These results suggest that HCV might affect cardiovascular events via pathways other than virus-induced metabolic modifications, such as IR and hypolipidemia, which may balance each other2,3. Although most HCV infections are curable using potent direct-acting anti-viral agents, not all HCV-associated cardiometabolic complications are reversible after viral clearance2. Free fatty acids and glycerol derived from visceral adipose tissue travel to the liver and stimulate lipoprotein synthesis and gluconeogenesis, respectively. Moreover, adipose tissue exerts important endocrine and immune functions through adipokines1,4. Thus, dissecting the relationship between HCV infection and adipokine alterations holds promise for preventing or treating HCV-associated cardiovascular complications. Among the adipokines, plasminogen activator inhibitor-1 (PAI-1), a single-chain glycoprotein with a molecular weight of 50 kDa that acts as a serine protease inhibitor (serpin)4, is a principal inhibitor of fibrinolysis due to its inactivation of plasminogen activators. PAI-1 is synthesized and secreted by ectopic fat depots, endothelial cells, hepatocytes, tumor cells and inflammation-activated cells and is present as a stored product in platelets4,5. 1
Liver Research Center, Division of Hepatology, Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 2Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 3Department of Cardiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 4Healthcare center, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 5Graduate Institute of Health-Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan. 6Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan. Correspondence and requests for materials should be addressed to M.-L.C. (email: [email protected]) Scientific Reports | 7:42503 | DOI: 10.1038/srep42503
1
www.nature.com/scientificreports/ The secretion of PAI-1 is stimulated by insulin, free fatty acids, atherogenic lipoproteins and chronic inflammation6. Consequently, PAI-1 levels are elevated during thrombotic, fibrotic, and cardiovascular events, as well as in the presence of hyperinsulinemia, hyperlipidemia, hypertension, non-alcoholic fatty liver disease and malignancy, especially those cancers with metastatic behavior and poor prognosis4–7. In contrast, PAI-1 secretion decreases with weight loss8. Additionally, PAI-1 works as an early response protein to modulate hepatocyte growth and differentiation9 and as a marker of senescence10. Previous studies have suggested that a single guanosine insertion/deletion (4 G/5 G) polymorphism in the promoter region and another nearby single nucleotide polymorphism (SNP) in the PAI-1 gene [SERPINE1 (7q22.1)], as well as several SNPs in chromosomes other than chromosome 7, are associated with circulating levels of PAI-111. These polymorphisms potentially increase the risk of IR and thrombosis formation12,13. With regard to the 4 G/5 G polymorphism, the 4 G allele is associated with moderately high PAI-1 levels. Furthermore, possible gene–environment interactions complicate this association, as differences in PAI-1 levels between individuals with the 4 G vs. the 5 G allele are more apparent in the presence of diseases that stimulate PAI-1 expression14. The dynamics of PAI-1 are particularly unpredictable in patients with chronic hepatitis C (CHC), as hyperfibrinolysis may occur due to elevated tissue-type plasminogen activator (tPA) levels5. In addition, HCV genotype-specific sustained virological response (SVR)-associated lipid alterations2,15 might complicate the transcriptional regulation of PAI-11,2. Given its complex gene/environmental coordination, the role of PAI-1 in cardiovascular risk for CHC patients remains elusive. Therefore, we sought to address the aforementioned enigmas by conducting a prospective study of PAI-1 levels in patients infected with various genotypes of HCV who had completed anti-HCV therapy. The study was adjusted for crucial confounders, including demographic characteristics as well as metabolic, liver, viral and genetic profiles.
Results
PAI-1-rs1799889 genotype correlated with pre-therapy PAI-1 levels in patients with CHC. The baseline (pre-therapy) characteristics of the patients with CHC are listed in Table 1. Patients with an SVR had lower levels of HCV RNA, homeostatic model assessment of insulin resistance (HOMA-IR) and brain natriuretic peptide (BNP) and a lower prevalence of cirrhosis and genotype 1 (G1) HCV infection. However, they had higher prevalences of G2 HCV infection and the interferon-λ3 (IFNL3) CC genotype and higher platelet counts than the nonSVR patients. No notable differences were found in PAI-1 levels between the patients with and without an SVR. Among the 5 PAI-1-associated SNPs evaluated in the patients with CHC, correlation tests showed that only the 4 G/5 G polymorphism of PAI-1-rs1799889 correlated with pre-therapy PAI-1 levels (Pearson’s correlation coefficient = 0.124, p = 0.044). Independent factors associated with pre-therapy PAI-1 levels in patients with CHC. Before anti-HCV therapy, as listed in Table 2, univariate and multivariate regression analyses showed that age, body mass index (BMI), platelet count and triglyceride (TG) levels as well as the PAI-1-rs1799889 4 G/4 G genotype were associated with PAI-1 levels. HCV genotype and white blood cell (WBC) count were associated with HCV RNA expression. HCV genotype, BMI, liver cirrhosis and IFNL3 genotype were associated with SVR. Independent factors associated with the longitudinal trend in PAI-1 levels in patients with CHC. Using the GEE method, the factors longitudinally affecting PAI-1 levels were identified and are listed
in Table 3. We found that sex, BMI, treatment, HOMA-IR, aspartate aminotransferase (AST) to platelet ratio index (APRI), platelet count, estimated glomerular filtration rate (eGFR), total cholesterol (TC), TG levels and PAI-1-rs1799889 and IFNL3-rs12979860 genotypes were independently associated with PAI-1 levels. Among these factors, the impacts of the categorical variables sex and PAI-1-rs1799889 and IFNL3-rs12979860 genotypes on PAI-1 levels were further elucidated and are presented in Fig. 1. The following observations were made: (1) Male patients consistently had higher PAI-1 levels than female patients throughout the therapy (Fig. 1A). (2) The positive impact of the PAI-1-rs 1799889 4 G/4 G genotype on PAI-1 level was most evident at the beginning of the therapeutic course and diminished as the course proceeded (Fig. 1B). (3) Patients with the IFNL3-rs12979860 CC genotype had higher PAI-1 levels than those with the non-CC genotype in the later part of the therapeutic course compared to the beginning (Fig. 1C).
Independent factors associated with post-therapy PAI-1 levels among patients with an SVR. Among the patients who achieved an SVR, univariate analysis and multivariate analysis confirmed that
sex (estimated β = 0.934, p = 0.031), age (estimated β = -0.074, p = 0.001), platelet count (estimated β = 0.026, p
OPEN
received: 01 September 2016 accepted: 11 January 2017 Published: 13 February 2017
Link between plasminogen activator inhibitor-1 and cardiovascular risk in chronic hepatitis C after viral clearance Ming-Ling Chang1,2, Yu-sheng Lin3,4, Li-Heng Pao5,6, Hsin-Chih Huang1 & Cheng-Tang Chiu1 The pathophysiological implications of plasminogen activator inhibitor-1 (PAI-1) in HCV infection remain obscure. This prospective study evaluated 669 HCV patients, of whom 536 had completed a course of anti-HCV therapy and had pre-, peri- and post-therapy measurements of various profiles, including PAI-1 levels. Multivariate analysis demonstrated, before anti-HCV-therapy, platelet count and PAI-1-rs1799889 genotype were associated with PAI-1 levels. Among patients with a sustained virological response (SVR, n = 445), platelet count was associated with PAI-1 level at 24 weeks posttherapy. GEE analysis showed that PAI-1-rs-1799889 and interferon-λ3-rs12979860 genotypes affected PAI-1 levels early and late in therapy, respectively. At 24 weeks post-therapy, higher lipid, brain natriuretic peptide, homocysteine and PAI-1 levels and PAI-1 activity were noted only in SVR patients compared with pre-therapy levels. Within 24 weeks post-therapy, 2.2% of the SVR (mean age: 57.8 yr; 8 smoking males; the 2 females had pre-therapy hypercholesteremia or cardiovascular family history of disease) and 0% of the non-SVR patients experienced a new cardiovascular event. Platelet counts consistently correlated with PAI-1 levels regardless of HCV infection. PAI-1-rs-1799889 and interferonλ3-rs12979860 genotypes mainly affected PAI-1 levels longitudinally. Within 24 weeks post-antiHCV therapy, the SVR patients showed increasing PAI-1 levels with accelerating cardiovascular risk, especially the vulnerable cases. Hepatitis C virus (HCV), a human pathogen responsible for acute and chronic liver disease, has variants that are classified into 7 major genotypes and has infected an estimated 170 million individuals worldwide1. HCV infection is believed to cause metabolic alterations, including steatosis, hypolipidemia, insulin resistance (IR), diabetes and obesity2. Despite the favorable lipid profile that results from HCV infection, many studies have shown that HCV infection unfavorably impacts cardiovascular events after adjusting for conventional risk factors2,3. These results suggest that HCV might affect cardiovascular events via pathways other than virus-induced metabolic modifications, such as IR and hypolipidemia, which may balance each other2,3. Although most HCV infections are curable using potent direct-acting anti-viral agents, not all HCV-associated cardiometabolic complications are reversible after viral clearance2. Free fatty acids and glycerol derived from visceral adipose tissue travel to the liver and stimulate lipoprotein synthesis and gluconeogenesis, respectively. Moreover, adipose tissue exerts important endocrine and immune functions through adipokines1,4. Thus, dissecting the relationship between HCV infection and adipokine alterations holds promise for preventing or treating HCV-associated cardiovascular complications. Among the adipokines, plasminogen activator inhibitor-1 (PAI-1), a single-chain glycoprotein with a molecular weight of 50 kDa that acts as a serine protease inhibitor (serpin)4, is a principal inhibitor of fibrinolysis due to its inactivation of plasminogen activators. PAI-1 is synthesized and secreted by ectopic fat depots, endothelial cells, hepatocytes, tumor cells and inflammation-activated cells and is present as a stored product in platelets4,5. 1
Liver Research Center, Division of Hepatology, Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 2Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan. 3Department of Cardiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 4Healthcare center, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 5Graduate Institute of Health-Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan. 6Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan. Correspondence and requests for materials should be addressed to M.-L.C. (email: [email protected]) Scientific Reports | 7:42503 | DOI: 10.1038/srep42503
1
www.nature.com/scientificreports/ The secretion of PAI-1 is stimulated by insulin, free fatty acids, atherogenic lipoproteins and chronic inflammation6. Consequently, PAI-1 levels are elevated during thrombotic, fibrotic, and cardiovascular events, as well as in the presence of hyperinsulinemia, hyperlipidemia, hypertension, non-alcoholic fatty liver disease and malignancy, especially those cancers with metastatic behavior and poor prognosis4–7. In contrast, PAI-1 secretion decreases with weight loss8. Additionally, PAI-1 works as an early response protein to modulate hepatocyte growth and differentiation9 and as a marker of senescence10. Previous studies have suggested that a single guanosine insertion/deletion (4 G/5 G) polymorphism in the promoter region and another nearby single nucleotide polymorphism (SNP) in the PAI-1 gene [SERPINE1 (7q22.1)], as well as several SNPs in chromosomes other than chromosome 7, are associated with circulating levels of PAI-111. These polymorphisms potentially increase the risk of IR and thrombosis formation12,13. With regard to the 4 G/5 G polymorphism, the 4 G allele is associated with moderately high PAI-1 levels. Furthermore, possible gene–environment interactions complicate this association, as differences in PAI-1 levels between individuals with the 4 G vs. the 5 G allele are more apparent in the presence of diseases that stimulate PAI-1 expression14. The dynamics of PAI-1 are particularly unpredictable in patients with chronic hepatitis C (CHC), as hyperfibrinolysis may occur due to elevated tissue-type plasminogen activator (tPA) levels5. In addition, HCV genotype-specific sustained virological response (SVR)-associated lipid alterations2,15 might complicate the transcriptional regulation of PAI-11,2. Given its complex gene/environmental coordination, the role of PAI-1 in cardiovascular risk for CHC patients remains elusive. Therefore, we sought to address the aforementioned enigmas by conducting a prospective study of PAI-1 levels in patients infected with various genotypes of HCV who had completed anti-HCV therapy. The study was adjusted for crucial confounders, including demographic characteristics as well as metabolic, liver, viral and genetic profiles.
Results
PAI-1-rs1799889 genotype correlated with pre-therapy PAI-1 levels in patients with CHC. The baseline (pre-therapy) characteristics of the patients with CHC are listed in Table 1. Patients with an SVR had lower levels of HCV RNA, homeostatic model assessment of insulin resistance (HOMA-IR) and brain natriuretic peptide (BNP) and a lower prevalence of cirrhosis and genotype 1 (G1) HCV infection. However, they had higher prevalences of G2 HCV infection and the interferon-λ3 (IFNL3) CC genotype and higher platelet counts than the nonSVR patients. No notable differences were found in PAI-1 levels between the patients with and without an SVR. Among the 5 PAI-1-associated SNPs evaluated in the patients with CHC, correlation tests showed that only the 4 G/5 G polymorphism of PAI-1-rs1799889 correlated with pre-therapy PAI-1 levels (Pearson’s correlation coefficient = 0.124, p = 0.044). Independent factors associated with pre-therapy PAI-1 levels in patients with CHC. Before anti-HCV therapy, as listed in Table 2, univariate and multivariate regression analyses showed that age, body mass index (BMI), platelet count and triglyceride (TG) levels as well as the PAI-1-rs1799889 4 G/4 G genotype were associated with PAI-1 levels. HCV genotype and white blood cell (WBC) count were associated with HCV RNA expression. HCV genotype, BMI, liver cirrhosis and IFNL3 genotype were associated with SVR. Independent factors associated with the longitudinal trend in PAI-1 levels in patients with CHC. Using the GEE method, the factors longitudinally affecting PAI-1 levels were identified and are listed
in Table 3. We found that sex, BMI, treatment, HOMA-IR, aspartate aminotransferase (AST) to platelet ratio index (APRI), platelet count, estimated glomerular filtration rate (eGFR), total cholesterol (TC), TG levels and PAI-1-rs1799889 and IFNL3-rs12979860 genotypes were independently associated with PAI-1 levels. Among these factors, the impacts of the categorical variables sex and PAI-1-rs1799889 and IFNL3-rs12979860 genotypes on PAI-1 levels were further elucidated and are presented in Fig. 1. The following observations were made: (1) Male patients consistently had higher PAI-1 levels than female patients throughout the therapy (Fig. 1A). (2) The positive impact of the PAI-1-rs 1799889 4 G/4 G genotype on PAI-1 level was most evident at the beginning of the therapeutic course and diminished as the course proceeded (Fig. 1B). (3) Patients with the IFNL3-rs12979860 CC genotype had higher PAI-1 levels than those with the non-CC genotype in the later part of the therapeutic course compared to the beginning (Fig. 1C).
Independent factors associated with post-therapy PAI-1 levels among patients with an SVR. Among the patients who achieved an SVR, univariate analysis and multivariate analysis confirmed that
sex (estimated β = 0.934, p = 0.031), age (estimated β = -0.074, p = 0.001), platelet count (estimated β = 0.026, p
